激动GPER-1受体对全脑缺血大鼠CA1区血脑屏障的保护作用及分子机制研究

发布时间：2018-02-10 19:06

  本文关键词： 全脑缺血模型 血脑屏障 GPER-1 VEGF-A　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:缺血性脑卒中是全球死亡率的第二大原因。系统研究脑缺血的治疗和机制需要一个稳定的、可复制性高的全脑缺血模型。尽管在许多研究模型中使用小鼠和沙鼠,但是可靠的全脑缺血大鼠模型也是实验所需要的,并且有待进一步改善。四血管阻断法是一种广泛使用的全脑缺血模型。手术需要灼烧大鼠寰椎的翼状孔,永久性阻断双侧椎动脉。但是,术者往往无法直视下电凝椎动脉使其完全闭塞。因此,建立成功的模型需要依赖观察大鼠的翻正反射和脑电图表现。本课题需要建立一种稳定的全脑缺血模型,为后续实验打好基础。血脑屏障是血浆与脑细胞之间的屏障,对中枢神经系统的内稳态和正常功能的维持至关重要。血脑屏障内皮细胞的功能和形态与非脑组织毛细血管内皮细胞截然不同。在许多神经系统疾病中,血脑屏障的完整性会遭到破坏,例如缺血性脑卒中。脑缺血对血脑屏障的损伤作用已被广泛研究。脑缺血缺氧会引起脑内一系列的分子变化,导致内皮细胞紧密连接破坏,血脑屏障通透性增加。许多研究证明脑缺血后雌激素可通过阻止血脑屏障的破坏从而保护脑组织。雌激素的脑保护作用是由雌激素受体介导的(ERα、ERβ、GPER-1)。有研究表明ERα和ERβ可介导雌激素对脑缺血后血管内皮细胞连接蛋白的保护作用。但是,GPER-1在脑缺血导致的血脑屏障损伤中的作用目前还不清楚。目的:1.探索大鼠椎动脉与颈椎的位置关系,寻找阻断大鼠椎动脉的最佳部位。2.建立稳定性高的新型全脑缺血模型,探索最佳缺血时间和最佳缺血部位。3.探索全脑缺血对去势大鼠海马CA1区血脑屏障以及GPER-1和VEGF-A表达的影响。4.探索激动GPER-1受体是否可通过降低VEGF-A表达来保护全脑缺血大鼠CA1区血脑屏障。方法:1.采用CTA、DSA、血管乳胶灌注法明确大鼠椎动脉走行以及与颈椎的解剖关系。寻找阻断大鼠椎动脉的最佳部位。2.建立新型全脑缺血模型。根据全脑缺血10、20、30分钟后大鼠的存活率,得出大鼠全脑缺血的最佳持续时间。随后进行灌注、固定、取脑,采用尼氏染色和TUNEL标记染色法观察脑缺血后大鼠各个脑区神经元的死亡情况,评估新型全脑缺血模型的稳定性。最后根据大鼠神经功能评分量表和胶带实验结果,观察全脑缺血大鼠的行为学变化,进一步评估模型对大鼠行为学的损害情况。3.采用Western blot实验技术观察全脑缺血20min后大鼠CA1区血脑屏障通透性的变化(Ig G漏出率)和连接蛋白(Occludin、Claudin-5)的表达变化。同时也对脑缺血后的CA1区GPER-1受体和VEGF-A蛋白的表达变化进行观察。4.采用Western blot和免疫荧光双标染色观察激动GPER-1受体对全脑缺血后大鼠CA1区血脑屏障通透性(Ig G漏出率)和连接蛋白(Occludin、Claudin-5)表达的影响。随后采用Western blot和免疫组织化学染色法探索激动GPER-1受体是否可通过降低VEGF-A表达来保护全脑缺血大鼠CA1区血脑屏障。结果:1.CTA、DSA、血管乳胶灌注法结果发现,在颈1-颈2横突间隙内椎动脉位于枢椎上关节突的外侧深面,并且不受上关节突遮挡,颈后路手术可以在显微镜直视下轻松阻断椎动脉。2.生存分析结果显示:10min全脑缺血大鼠术后7天的生存率达93%;20min全脑缺血大鼠术后7天的生存率达80%;30min全脑缺血大鼠术后7天的生存率仅为50%。制定20min为新型全脑缺血模型的最佳缺血时间。3.尼氏染色结果显示:20min全脑缺血大鼠CA1区神经元数量在术后第1天无统计学差异,但在术后第3天出现明显下降。在随后的3-7天中,CA1区神经元数量没有进一步下降。制定CA1区为新型全脑缺血模型的最佳缺血部位。4.胶带实验和TUNEL染色结果证明全脑缺血大鼠在术后7天内皮层区和纹状体区神经元均存在凋亡。新型全脑缺血模型可以导致大鼠运动整合能力相关脑区神经元损伤,并且这些神经元的损伤可以通过胶带实验进行观察评价。5.Western blot检测结果显示:全脑缺血大鼠CA1区Occludin和Claudin-5蛋白含量在缺血后6小时开始降低,24小时明显降低。后续实验可以采用全脑缺血后24小时为观察时间点。6.Western blot和免疫荧光双标染色结果显示:激动GPER-1受体可以阻止缺血后OVX大鼠CA1区连接蛋白表达(Occludin和Claudin-5)的下降。另外还发现给予G-1激动剂后全脑缺血OVX大鼠CA1区VEGF-A表达明显下降。结论:1.新型全脑缺血模型可以稳定的造成大鼠海马CA1区神经元迟发性死亡,并引起大鼠神经功能出现异常。20min的全脑缺血为最佳缺血时间,CA1区为最佳缺血部位。2.新型全脑缺血模型可以导致大鼠海马CA1区血脑屏障的破坏,并且在缺血后24小时,CA1区血脑屏障破坏最为明显。3.激动GPER-1受体可在脑缺血早期起到稳定血脑屏障通透性的作用。激动GPER-1受体可以通过减少VEGF-A的表达来稳定缺血CA1区血脑屏障的通透性。
[Abstract]:Background: ischemic stroke is the second leading cause of mortality worldwide. The treatment and mechanism of cerebral ischemia research requires a stable, reproducible model of global cerebral ischemia is high. Although the use of mice and gerbils in many models, but the cerebral ischemia rats model is reliable experiment. And it needs to be further improved. The four vessel occlusion method is a widely used model of global cerebral ischemia. The rats of hole surgery requires cautery of atlas, the permanent occlusion of bilateral vertebral artery. However, patients are often unable to open electrocoagulating the complete occlusion of the vertebral artery. Therefore, a successful model relies on turning reflection and electroencephalogram of rats were observed. The need to establish a stable model of global cerebral ischemia, to lay a good foundation for the subsequent experiments. The blood-brain barrier is between plasma and brain cells of the central nervous system barrier. Critical to maintain homeostasis and normal function. The endothelial cells of blood brain barrier function and morphology and non brain capillary endothelial cells are quite different. In many nervous system diseases, the integrity of the blood-brain barrier will be destroyed, such as ischemic stroke. The damage effect of cerebral ischemia on blood brain barrier in the brain has been widely studied. Hypoxia ischemia will cause a series of molecular changes in the brain, leading to endothelial cell tight junction damage, increase the permeability of blood brain barrier after cerebral ischemia. Many studies have shown that estrogen can prevent the destruction of the blood-brain barrier to protect the brain. Cerebral protective effect of estrogen is mediated by estrogen receptors (ER alpha, ER beta. GPER-1). Studies have shown that ER alpha and ER beta can mediate the protective effects of estrogen on vascular endothelial cell junction protein after cerebral ischemia. However, blood brain barrier damage in cerebral ischemia caused by GPER-1 The role is unclear. Objective: To explore the relationship between the 1. position and cervical vertebral artery of rats, to find.2. best position to block rat vertebral artery to establish high stability model of cerebral ischemia, to explore the best time and the best part of ischemia.3. ischemia cerebral ischemia exploration of ovariectomized rat hippocampal CA1 region blood brain barrier and the expression of GPER-1 and VEGF-A.4. to explore whether activation of GPER-1 receptor can reduce the expression of VEGF-A to protect cerebral ischemia area CA1 blood brain barrier. Methods: 1. using CTA, DSA, vascular latex perfusion clear rat vertebral artery and cervical spine anatomy and.2.. Looking for the best position to block rat the vertebral artery to establish a new model of global cerebral ischemia. According to the survival rate of cerebral ischemia in rats after 10,20,30 minutes, the optimal duration of cerebral ischemia rats. Then perfusion fixed, brain was removed by Nissl staining Observation of cerebral ischemia and TUNEL color marker staining in various brain regions of neurons in rats after death, assessing the stability of whole brain ischemia model model. Finally, according to the neural function of rats and tape scale experimental results, to observe the pathological change of cerebral ischemia in rats, further evaluation model by changes in rats CA1 in the permeability of blood brain barrier after cerebral ischemia 20min observation of Western blot experimental technique on the behavior of rats damage.3. (Ig G leakage rate) and connexin (Occludin, Claudin-5). The changes of the expression and expression of CA1 after cerebral ischemia GPER-1 receptor and VEGF-A protein were observed by Western.4. blot and immunofluorescence staining were used to observe the activation of GPER-1 receptor on CA1 blood brain barrier permeability in rats after cerebral ischemia (Ig G leakage rate) and connexin (Occludin, Claudin-5) on the expression of the Wes. Tern blot and immunohistochemical staining to explore whether activation of GPER-1 receptor can reduce the expression of VEGF-A to protect cerebral ischemia area CA1 blood brain barrier. Results: 1.CTA, DSA, vascular latex perfusion showed that the lateral aspect in carotid 1- cervical transverse process of the 2 gap within the vertebral artery is located in the upper joint axis process, and is not affected by the superior articular process of cervical posterior occlusion in the surgery under the microscope can easily block the vertebral artery.2. survival analysis showed: 10min cerebral ischemia rats 7 days after operation, the survival rate was 93%; 20min cerebral ischemia rats 7 days after operation, the survival rate was 80%; the best time of ischemia.3. 7 rats in the postoperative survival rate is only 50%. 20min for making model of whole brain ischemia cerebral ischemia 30min Nissl staining results show that: the number of neurons in CA1 area of rats with cerebral ischemia 20min on the first day after operation there was no significant difference, but on the third day after operation significantly Decreased. In the subsequent 3-7 days, the number of CA1 neurons decreased. No further develop CA1 area for the whole brain ischemia model best ischemia.4. tape test and TUNEL staining results showed that cerebral ischemia rats after 7 days in the cortex and striatum neuron apoptosis. There are new models of ischemia the brain can lead to neuronal damage in rat brain areas related to motion integration capabilities, and these neurons can observe the evaluation of.5.Western blot showed that by tape experiments: whole brain Occludin and Claudin-5 protein in ischemic rats CA1 content began to decrease at 6 hours after ischemia, 24 hours can be significantly reduced. The subsequent experiments using whole brain 24 hours after ischemia for observation time point.6.Western blot and immunofluorescence staining showed that activation of GPER-1 receptor can prevent OVX after ischemia in rat CA1 region binding protein table Da (Occludin and Claudin-5). It is also found that the decrease of cerebral ischemia in OVX rats of CA1 VEGF-A expression was significantly decreased after the administration of G-1 agonists. Conclusion: 1. new models of global cerebral ischemia can cause stability in rat hippocampal CA1 neurons delayed neuronal death, and cause the neurological function of rats with abnormal.20min full cerebral ischemia is the best time of ischemia, the CA1 district was the best site of ischemia.2. new model of global cerebral ischemia can lead to hippocampal CA1 region of rat blood brain barrier damage, and in 24 hours after ischemia, the most obvious.3. activation of GPER-1 receptor in the early stage of cerebral ischemia to stabilize the BBB permeability to blood brain area CA1 barrier damage. Activation of GPER-1 receptor can stabilize CA1 ischemia area of blood brain barrier permeability by decreasing the expression of VEGF-A.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3
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本文编号：1501220